Tn3-like structures co-harboring of blaCTX-M-65, blaTEM-1 and blaOXA-10 in the plasmids of two Escherichia coli ST1508 strains originating from dairy cattle in China

The purpose of this study was to determine the level of horizontal transmission of the blaCTX-M-65 gene and the role of its associated mobile genetic elements (MGEs) in the bovine-derived Escherichia coli. After PCR identification, two plasmids carrying blaCTX-M-65 were successfully transferred to the recipient E. coli J53 Azr through conjugation assays and subsequently selected for Whole-Genome sequencing (WGS) analysis. The resistance profiles of these two positive strains and their transconjugants were also determined through antimicrobial susceptibility tests. Whole genome data were acquired using both the PacBio sequencing platform and the Illumina data platform. The annotated results were then submitted to the Genbank database for accession number recording. For comparison, the genetic environment of plasmids carrying the resistance gene blaCTX-M-65 was mapped using the Easyfig software. WGS analysis revealed Tn3-like composite transposons bearing blaCTX-M-65, blaTEM-1, and blaOXA-10 in the IncHI2-type plasmids of these two E. coli ST1508 strains. A phylogenetic tree was generated from all 48 assembled E. coli isolates blaCTX-M-65, blaTEM-1, and blaOXA-10 from the NCBI Pathogen Detection database with our two isolates, showing the relationships and the contribution of SNPs to the diversity between genetic samples. This study suggests that the transmissibility of blaCTX-M-65 on Tn3-like composite transposons contributes to an increased risk of its transmission in E. coli derived from dairy cattle.


Introduction
Since the 1980s, extended-spectrum β-lactamases (ESBL)-producing E. coli has spread globally, thus becoming the primary cause of nosocomial and community-acquired infections such as gastrointestinal and extra-intestinal infections which majorly includes urinary tract infections (UTIs) [1,2].Most of E. coli strains are extended beta-lactamase producing and identified as multidrug resistant (resistance to penicillin, cephalosporin's, and monobactems) due to the hydrolyzaiton of betalactam ring.The most prevalent ESBL families include CTX-M, TEM, OXA and SHV, with CTX-M gradually emerging as the dominant variant [3][4][5].The expression of CTX-M family genes is often associated with increase drug resistance to multiple beta-lactam antibiotics which restrict the more options for therapeutic purposes leading to the treatment options are severely limited [6].In addition, CTX-M genes have been widely identified in E. coli isolated from multiple sources such as humans, livestock, companion animals, food products, and environment, raising significant concerns [1,6].
The CTX-M genes are rarely found on chromosomes, instead encoded on mobile genetic elements (MGEs), usually on plasmids.Plasmid located CTX-M genes has a strong frequency of horizontal transfer and their increased dissemination within one health care (humans, animals, and environment) [7,8].MGEs located on plasmids can widely spread antibiotic resistance genes (ARGs) [9].It follows that Enterobacteriaceae have adopted a crucial strategy to capture, stabilize and disseminate new genetic elements during the acquisition and evolution of ESBL enzymes [6].The current epidemiological scenario suggests that the transmission pattern of the CTX-M enzyme may be allodemic rather than epidemic.In other words, the widespread prevalence of CTX-M enzyme is not due to the proliferation of specific clones, but rather to the proliferation of multiple specific clones or MGEs [10].
Co-transmission of CTX-M-65 with other ESBL-ARGs in E. coli have also been reported more frequently, ultimately leading to an increased risk of livestock-to-human transmission and global multidrug resistance (MDR) through horizontal gene transfer (HGT) [11][12][13][14].Note that bla CTX-M-65 gene, with an elevated detection rate, has been reported in food animals [15,16].Food-producing chickens carrying extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), including CTX-M-65-producing Escherichia coli have posed a potential threat to human and animal health [17].As a result, there is an increased risk of transmission of these genes from food animals to humans.In this study, we characterized and investigated the horizontal transfer mechanism of bla CTX-M-65 from bovine origin through whole genome sequencing and conjugation assay.

Strain isolation
73 non-repetitive fecal samples were collected from dairy cattle in the Xinjiang region of China in 2018.All samples were collected in sterile containers, transported to the laboratory at 6 °C ± 2 °C, and processed immediately for further assays.All isolates were selected on MacConkey agar plates (Huankai, China) and identified by MALDI-TOF-MS and 16 s rRNA sequencing using universal primers F (5'-GAG CGG ATA ACA ATT TCA CAC AGG -3') and R (5'-CGC CAG GGT TTT CCC AGT CAC GAC -3').All E. coli strains were stored in our laboratory after identification.
CTX-M-65-positive strains were screened for the presence of bla CTX-M-9 group using PCR methods with primers obtained from the article [18].PCR products were confirmed using Sanger sequencing.

Conjugation assay and determination of conjugation frequency
Conjugation assays were performed among 2 isolates.To determine the transferability of the resistance genes, filtered broth mating was performed using E. coli J53 Az r as the recipient [19].The donor-recipient ratio was 1:1 using Mueller-Hinton medium (MHA, Huankai, Guangzhou, China) supplemented with cefotaxime (2 μg/mL) and sodium azide (200 μg/mL) as the selective medium.Positive clones of transconjugants were screened by PCR (bla CTX-M-9 primers, F: 5'-ATG GTG ACA AAG AGA GTG CAAC-3' , R: 5'-TTA CAG CCC TTC GGC GAT G-3'), Sanger sequencing and antimicrobial susceptibility testing.Transfer frequencies were calculated as the number of transconjugants per recipient.

WGS sequencing and analysis
The total genomic DNA was extracted with the SDS method [20].The harvested DNA was detected by the agarose gel electrophoresis and quantified by Qubit ® 2.0 Fluorometer (Thermo Scientific).The whole genome of the isolate was sequenced on PacBio Sequel single-molecule real-time (SMRT) sequencing platform (Novogene Bioinformatics Technology Co., Beijing, China) with 400 bp paired-end libraries.It was preliminarily assembled with SMRT Link v5.0.1.The assembled data were optimized by Illumina reads using Burrows-Wheeler Aligner (BWA).
To confirm the phylogenetic relationship based on the single nucleotide polymorphisms (SNPs) present in the genomes carrying the bla CTX-M-65 gene, the two E. coli strains in this study and the 48 E. coli strains downloaded from the NCBI Pathogenic Detection were analyzed using CSI Phylogeny version 1.4 (10 reads of minimal depth at SNP positions, 10% minimal relative depth at SNP positions, 10 bp of minimal distance between SNPs, minimal SNP quality of 30, minimal read mapping quality of 25, and a minimal Z-score of 1.96 [22].The phylogenetic tree was visualized using FigTree version v1.4.4.

Phylogenetic analysis
The phylogenetic analysis was performed using the method of Kaas [23].A phylogenetic tree was generated using E. coli ATCC25922 as the reference genome for all assembled E. coli isolates carrying bla CTX-M-65 , bla TEM-1 , and bla OXA-10 from the NCBI Pathogenic Detection database.To be specific, a total of 50 E. coli isolates carrying these ARGs were collected from different regions of the world and isolated from various sources.

Results and discussion
A total of 108 E. coli strains were isolated on MacConkey agar from dairy cow and confirmed by 16 s rRNA and MALDI-TOF-MS.Among the 108 E. coli strains, three strains were identified positive for bla CTX-M-65 gene by PCR method in our previous study [24].In two strains, bla CTX-M-65 gene was successfully transferred through conjugation which were further characterized for antimicrobial susceptibility testing, acquired ARGs, and related the genetic environment through WGS analysis.The information about the origin and source of the isolates is shown in Table 1.
The Kirby-Bauer disk diffusion method was used to evaluate the drug resistance profile against multiple antibiotics.The results showed that both of the strains were multidrug-resistant (MDR) and resistant to at least nine antibiotics.Both of the isolates showed resistance to cefalotin (KF), cefotaxime (CTX), tetracycline (TE), chloramphenicol (C), ampicillin (AMP), doxycycline (DO), sulbactam-ampicillin (SAM) and strimethoprimsulfamethoxazole (SXT) (Table 1).A previous conducted by researchers also identified MDR E. coli strains isolated from dairy origin which is consistent to the current findings [25].The ESBL-producing E. coli also displayed a wide spectrum antibiotic resistance to other commonly used antibiotics at clinical and livestock industry such as β-lactam, tetracycline and chloramphenicol and trimethoprim-sulfamethoxazole which results in limited treatment options and higher rate of therapeutic failures in infections caused by ESBL-producing E. coli.The conjugation assay revealed that two plasmids carrying CTX-M-65 gene were successfully transferred to the recipient E. coli J53 (Sodium Azide resistance) indicating the presence of the target gene on a mobile plasmid.The conjugation frequencies of XJ6-3-J53 and XJ34-J53 carrying CTX-M-65 gene were noted to be 1.9 × 10 -6 and 6.9 × 10 -5 conjugants per recipient respectively which is in consistent with a previous study [26].
Both of the strains were sent for WGS and characterized for plasmid replicon typing, serotyping, MLST, acquired ARGs, and phylogenetic relationship using CGE website.Plasmid replicon typing revealed that bla CTX- M-65 gene was localized on IncHI2 plasmid type in both strains along with other replicon types such as IncI1, IncF, and IncA/C2 plasmids (Table 1) which are previously known as dominant plasmid replicon types [11,27].
The MLST revealed that both CTX-M-65-positive strains belongs to ST1508.Surprisingly, after searching the Pathogen Detection database, none of the CTX-M-65 positive strain was ST1508 which indicate the emergence of a novel ST1508 in this study.Moreover, both XJ6.3 and XJ34 strains were categorized under O172:H45 serotype which belongs to toxin producing E. coli.E. coli O172 producing Shiga toxin type 2 has been reported from food and water sources commonly associated with illnesses [28,29].XJ6.3 and XJ34 have certain degree of pathogenicity and resistance, therefore their possible transmission need greater attention at the right time.
Two plasmids carrying CTX-M-65 gene were successfully transferred to the recipient E. coli J53 Azr according to conjugation assays, indicating the presence of the CTX-M-65 gene on a plasmid.The conjugation frequencies of J53XJ6-3 and J53XJ34 carrying CTX-M-65 gene were 1.9 × 10 -6 and 6.9 × 10 -5 , respectively.The transconjugative frequencies of conjugants of harboring CTX-M-65 genes were 10 -5 ~ 10 -6 in consistent with the additional research of CTX-M-65 on IncHI2 [26].
The mobile genetic elements such as IS903 and Tn3 are the most prevalent insertions and transposons which associated with bla CTX-M-65 which is consistent with our findings that bla CTX-M-65 gene was surrounded by IS903 and Tn3-like mobile elements [31].The Tn3 belong to bacterial transposons group which are involved in transposition function and mediation of gene re-assortment, which make it conducive for horizontal gene transfer in gram-positive and negative bacteria [32].In addition, the res site provides more clarity on the Tn3-like transposon formed by transduction and integration to transmit ARGs.The presence of these elements in the genome of bla CTX-M-65 -positive strains mediates the mechanism of genes mobilization and increase the risk of horizontal transmission through multiple mobile elements [33].The phylogenetic relationship of two strains harboring bla CTX-M-65 gene and other strains retrieved from NCBI database were done by CSIPhylogeny tool based on SNPs.Both strains of present study shared 14 SNPs with each other indicating much homology with each other.However, both strains shared lower homology with other 48 strains retrieved from NCBI based on SNP differences ranging from 26,834 to 35,699.In addition, SNP analysis showed that SAMD00532258 strain were having highest SNPs, between 58,737 and 53,612.The number of SNPs in the majority of the strains carrying three ARGs were ranged from 847 to 58,737.The phylogenetic tree distinguished the two strains of this study and 48 from NCBI into 4 major clades (Fig. 2).Clade 1 consist of 20 strains, one from USA and left from China isolated from chicken, swine, duck, Manis javancia, human and water sources.Clade 2 were containing 15 strains from four different countries and sources.Clade 3 were consisting of two strains characterized in present study harboring bla CTX-M-65 under separate sub-clade and 13 other strains isolated from humans and animals in China.The phylogenetic analysis revealed that there was a high degree of genetic homology between the two strains (XJ6.3 and XJ34), marked with green box of this study, and lower homology with other strains.The aforementioned genetic differences from phylogenetic analysis indicate that these genes are more likely to be transmitted horizontally by plasmids rather than clonal dissemination.

Conclusion
The WGS analysis obtained a complete map of the genetic environment of CTX-M-65 in both isolates through the Pacbio sequencing platform and Illumina data platform.The CTX-M-65 positive E. coli isolates derived from dairy cattle and was to show co-transmission of bla CTX-M-65 with other ESBLs genes (bla TEM-1 , bla OXA-10 ) on the IncHI2 plasmid.A genetic environmental analysis of the CTX-M-65 gene revealed the presence of Tn3-like transposons and IS903 in both strains, possibly contributing to the mobilization of those ARGs.Genetic differences from phylogenetic analysis display showing the relationships and the contribution of SNPs to the diversity between genetic samples and co-transmission of these genes is more likely to occur through plasmids than through clones.Also, CTX-M-65 was derived from normal dairy cattle in this study, and it is still not absolutely certain that the source of CTX-M-65 is food dairy cattle.Finally, this study provides evidence of the need for appropriate measures to minimize the spread of MDR bacteria in dairy cattle samples.

Table 2
Characterization of plasmids carrying bla CTX-M-65